This invention relates to a DNA base sequencer, or an apparatus for determining the base sequences of DNA. More particularly, this invention relates to an apparatus with which the base sequences of DNA can be determined by fluorescent labelling in an efficient and rapid manner.
Gel electrophoresis is practiced extensively as a technique for determining the base sequences of DNA and other proteins. Conventionally, the sample to be subjected to electrophoresis is labelled with a radioisotope for analysis but this method has had the problem of being painstaking and time-consuming. Furthermore, the use of radioactive substances always calls for utmost safety and management and analysis cannot be performed in areas other than facilities that clear certain regulations. Under the circumstances, a method that uses fluorophores to label the sample and which detects fluorescences as emitted upon irradiation with light is being reviewed.
In this method, fluorophore-labelled DNA fragments are caused to migrate through a gel and a light excitation portion and a photodetector are provided for each electrophoresis track in an area 15-20 cm below the start point of electrophoresis. The DNA fragments are assayed as they pass through the line connecting the light excitation portion and the photodetector. A typical procedure of the method is described below. First, using as template the DNA chain to be determined for its base sequence, DNAs of various lengths with known terminal base species are replicated by a method involving an enzymatic reaction (the dideoxy method). Then, the replicated DNAs are labelled with a fluorophore. Stated more specifically, there are prepared a group of adenine (A) fragments, a group of cytosine (C) fragments, a group of guanine (G) fragments and a group of thymine (T) fragments, all being labelled with a fluorophore. A mixture of these fragment groups is injected into separate lane grooves in an electrophoretic gel and, thereafter, a voltage is applied at opposite ends of the gel. Since DNA is a chained polymer with negative charges, it will move across the gel at a rate in inverse proportion to its molecular weight. The shorter the DNA chain (the smaller its molecular weight), the faster will it move and vice versa; this is the principle behind the fractionation of DNA by molecular weight.
Japanese Laid-Open Patent Application (Kokai) No. 21556/1988 teaches a DNA base sequencer that is adapted in such a way that a line on the gel in an apparatus for electrophoresis at which laser light is applied and the direction in which photodiodes are arranged are both perpendicular to the direction in which DNA fragments migrate in the apparatus. The setup of this apparatus is shown schematically in FIG. 6. An electrophoresis plate 74 comprises a gel (typically a polyacrylamide gel) held between two glass plates. The electrophoresis plate has an overall thickness of up to about 10 mm but the thickness of the gel electrolyte layer itself is less than about 1 mm. The upper end of the gel electrolyte layer is comb-shaped and located slightly below the upper end of the plate 74. Fluorophore-labelled DNA fragments to be assayed are injected into grooves 75 corresponding to the teeth of the comb.
In the apparatus shown in FIG. 6, a laser beam emitted from a light source 70 is reflected by a mirror 72 and launched horizontally from one side of the plate 74 at a predetermined point on the gel. As the fluorophore-labelled DNA fragments migrating through the gel pass through the irradiated region, they will fluoresce successively. The horizontal position of fluorescence emission tells the species of a particular terminal base, the time difference from the start of migration tells the length of a particular fragment, and the emission wavelength identifies the sample under assay. The fluorescence from each electrophoresis track is condensed by a lens 78 to focus at a light-receiving area in an image intensifier 80. The received signal is amplified and converted to an electric signal in a photodiode array 84 for the purpose of various measurements. The results of measurements are processed with a computer so that the sequences of the individual DNA fragments are calculated to determine the base sequence of the DNA at issue.
The apparatus shown in FIG. 6 uses an image intensifier camera in the light-receiving optics. The image intensifier camera is not only very expensive but also comparatively large as an optical device. Under the circumstances, an apparatus of the design shown in FIG. 7 was developed, which had an individual fluorescence detecting means provided in the detection position of each electrophoresis track 88. Each fluorescence detecting means consisted of a filter 100, a condensing lens 110 and a light-receiving device 120. Filter 100 is provided for rejecting the excited light and the background light so that fluorescence is transmitted selectively. Condensing lens 110 is used for insuring that the filtered fluorescence is focused at the light-receiving plane of the device 120. The light-receiving device 120 is typically a photodiode composed of a silicon crystal having a pn junction.
A problem with the apparatus shown in FIG. 7 is that electrophoresis tracks are sensitive to external factors including temperature conditions, temperature profile and gel dissolution, so that the tracks deviate progressively from the linear state and as they run downward, the tracks become curved progressively until they occasionally shift either to the right or to the left. This shift of electrophoresis tracks is generally called "smiling". If smiling occurs, the position where fluorescence is emitted in response to the entrance of laser excited light into the DNA sample will deviate from the position where the fluorescence detecting means is provided; as a result, the efficiency of fluorescence detection drops and the precision with which the base sequences of DNA can be determined will decrease accordingly.
A further problem with the apparatus shown in FIG. 7 originates from the use of the condensing lens in each fluorescence detecting means. That is, the reduced fluorescent image causes an interference between adjacent electrophoresis tracks and it occasionally becomes difficult to achieve full separation of the fluorescent image waveform formed in one electrophoresis track from the waveform formed in an adjacent track, whereby the resolving power of the apparatus is lowered. To maintain a reasonable resolving power, the distance between electrophoresis tracks has to be increased but then the number of samples that can be analyzed at a time decreases, so does the throughput of the analysis.